The present invention disclosed herein relates to complexes of the Nlk1 protein with other proteins, in particular, complexes of the Nlk1 protein with the following proteins: TrkA, protein phosphatase-1xcex1, 14-3-3xcex5, xcex1-tropomyosin, vimentin, p0071, Ini-1, IP-1 (an intermediate filament associated protein), IP-2 (a tropomyosin homolog protein), IP-3 (a ubiquitin hydroxylase homolog protein), IP-4 (a collagen homolog protein) and IP-5 (a tropomyosin homolog protein). In addition, the present invention relates to the production of antibodies to the aforementioned Nlk1 protein complexes, and their use in, inter alia, screening, diagnosis, prognosis and therapy. The present invention further relates to the IP-1, IP-3, IP-4, and IP-5 genes and proteins, as well as derivatives, fragments, analogs and homologs, thereof.
It is a well-established tenet in molecular biology that loss of control of cell proliferation may lead to severe diseases and disorders (e.g., neoplasia). Hence, the elucidation of the intricacies of the cell-cycle, and its deregulation during oncogenesis, will provide novel opportunities in the prophylactic, diagnostic and therapeutic management of cancer and other proliferation-related diseases. A better understanding of the cell-cycle could be achieved by the elucidation of the interactions of the various protein complexes, whose levels and biological activities are regulated through the cell-cycle. The identification and classification of these protein complexes will be useful in the development of treatment modalities and assays for various pathological processes including, but not limited to, hyperproliferative disorders (e.g., tumorigenesis and tumor progression), as well as other related genetic disorders.
It should be noted that the citation of a reference herein should not be construed as an admission that such is prior art to the present invention.
(1) The Nlk1 Protein
The Nlk1 protein (GenBank Acc. No. U11050) is a human homolog of the filamentous fungus Aspergillus nidulans mitotic regulator, NIMA kinase. The Nlk1 protein is a 48 Kdal serine/threonine-specific kinase which plays a key role in cell-cycle events leading to the onset of mitosis. The protein levels and activity of Nlk1 protein are regulated through the cell-cycle (see e.g., Schultz, et al., 1994. Cell Growth Diff. 5:625-632; Fry, et al., 1995. J. Biol Chem. 270:12899-12905). Analysis of the biochemical properties and in vitro substrate-specificity of Nlk1 protein have revealed striking similarities, but also some differences, between human Nlk1 protein and the fungal mitotic regulator, NIMA kinase. See e.g., Fry, et al., 1995. J. Biol. Chem. 270:12899-12905. Nlk1 protein is expressed during specific stages of the cell-cycle; with low levels of expression during mitosis (M) phase and in early gap phase (G1), and expression peaking during the DNA-synthesis (S) and late gap phase (G2) to reach a plateau in late G2 and M-phase. See e.g., Id. Accordingly, Nlk1 protein may function during an earlier phase of the cell-cycle than NIMA kinase, which displays maximum protein and activity levels during mitosis. See e.g., Osmani, et al., 1991. EMBO J. 10:2669-267; Pu, et al., 1995. J. Biol Chem. 270:18110-18116. The Nlk1 protein is associated with the centrosome throughout the cell-cycle, including all stages of mitosis, independent of microtubules. See e.g., Fry, et al., 1998. EMBO J. 17:470-481. Thus, one biological function of Nlk1 protein relates to the centrosome cycle.
In certain aspects, the Nlk1 protein appears to function in a similar manner to NIMA kinase. See generally, Pu, et al., 1995. J. Biol. Chem. 270:18110-18116. The activity of NIMA kinase is essential for the progression of cells into mitosis, and the full activation of the NIMA kinase depends on the cyclin-dependent kinase, CDC2. Both NIMA and CDC2 kinases have been demonstrated to be required for the progression from G2 to mitosis in Aspergillus, and following this cell-cycle progression, both kinases are rapidly degraded. See e.g., Pu and Osmani, 1995. EMBO J. 14:995-1003. Recent experimental evidence has demonstrated that the Aspergillus NIMA serine/threonine kinase is not only required for mitosis, in cooperation with CDC2, but is also implicated in chromatin condensation. See e.g., Lu and Hunter, 1995. Cell 81:413-424; Pu and Osmani, 1995. EMBO J. 14:993-1003. Additionally, the Nlk1 protein may also be involved in other events of meiosis including, but not limited to, chromosomal condensation. See e.g., Rhee and Wolgemuth, 1997. Development 124:2167-2177.
As previously discussed, CDC2 is responsible for the phosphorylation of the Aspergillus NIMA kinase (see Fry and Nigg, 1995. Curr. Biol. 5:1122-1125), interestingly, however, mammalian Nlk1 protein lacks the putative CDC2 phosphorylation sites. While Nlk1 protein has been shown to be active as a serine/threonine kinase, the precise sites which are phosphorylated by Nlk1 protein have not yet been mapped. Recently, a synthetic peptide possessing the amino acid sequence, IRRLSTRR, was found to be phosphorylated exclusively on serine residues, thus tending to suggest that basic amino acid residues may contribute to substrate recognition by Nlk1 protein. See e.g., Fry, et al., 1995. J. Biol. Chem. 270:12899-12905.
The Nlk1 protein possesses marked sequence homology with NIMA kinase, with the two kinases sharing a 48% sequence identity over their catalytic domains located at the amino-terminus. See e.g., Schultz, et al., 1994. Cell Growth Diff. 5:625-635. Another conserved feature of the NIMA-related kinase family is the position of an insertion (maximal 25 amino acids) between two kinase the subdomains, VIA and VIB. Interestingly, the carboxyl-terminal extensions display virtually no primary sequence conservation among the different members of the NIMA-related kinase family. However, the beginning of the highly basic carboxyl-terminal, non-catalytic extension contains a conserved secondary structural motif (i.e., a coiled-coil) in both NIMA kinase and Nlk1 protein, which functions in the degradation of NIMA kinase during the M-phase. Coiled-coil motifs are often found to be involved in protein-protein interaction, and such secondary structural motifs may have been conserved as a direct result of their putative biological function(s). See e.g., Fry and Nigg, 1997. Meth. Enzym. 283:270-282. The carboxyl-terminal non-catalytic domain within human Nlk1 protein are markedly shortened, and it is interesting to note that several putative CDC2 phosphorylation sites found within the carboxyl-terminal domain of NIMA, are not found to be present in the much shorter extension of Nlk1 protein.
The Aspergillus NIMA kinase interacts with CDC2-kinase (see e.g., Osmani, et al, 1994. J. Cell Sci. 107:1519-1528) and with the human Pin1 protein, a peptidyl-prolyl-isomerase (see e.g., Lu, et al, 1996. Nature 380:544-547). Heretofore the present invention, no interacting proteins have been described for the human NIMA kinase homologue Nlk1 protein. The Nlk1 protein differs from NIMA kinase in that: (i) Nlk1 protein activity peaks in the cell-cycle""s S/G2 phase, and not in mitosis; (ii) Nlk1 protein lacks the putative CDC2 phosphorylation sites found in NIMA kinase and (iii) Nlk1 protein lacks the carboxyl-terminal PEST sequences found in NIMA kinase. Hence, the exact biological function(s) of the Nlk1 protein have yet to be elucidated, although along with the other members of the Nek family, it almost certainly functions in some form of cell-cycle mediation and control.
(2) Nlk1 protein-Interacting Proteins
The Nlk1 protein, a serine/threonine-specific kinase, plays an important role in cell-cycle events which lead to the onset of mitosis. Furthermore, Nlk1 protein is also involved in chromosome condensation and thus, in events of meiosis. Despite these aforementioned roles, heretofore the present invention, there has been no quantitative data regarding the interaction of the Nlk1 protein with other cellular proteins.
It is a well-established tenet in molecular biology that loss of control of cell proliferation may lead to severe diseases and disorders (e.g., neoplasia). As Nlk1 protein is involved in regulation of cell proliferation due to its role in the cell-cycle (mitosis and meiosis), it is likely that Nlk1 protein interacts with numerous cellular proteins to promote the profound structural reorganizations which accompany the entry of cells into, for example, mitosis. The elucidation of the intricacies of the cell-cycle, and its deregulation during oncogenesis, will provide novel opportunities in the prophylactic, diagnostic and therapeutic management of cancer and other proliferation-related diseases.
A better understanding of the cell-cycle could be achieved by the elucidation of the interactions of Nlk1 protein with other cellular proteins. As both the levels and biological activity of the Nlk1 protein are regulated through the cell-cycle, it is highly likely that this kinase will perform via protein-protein interactions. Although, as previously discussed, nothing is known within the prior art regarding Nlk1 protein""s interaction with other cellular proteins. Therefore, what is lacking within the prior art, is the identification of protein complexes between Nlk1 protein and other cellular proteins and/or polypeptides. The identification of these protein complexes will be useful in the development of treatment modalities and assays for various pathological processes including, but not limited to, hyperproliferative disorders (e.g., tumorigenesis and tumor progression), as well as other related genetic disorders.
(A) Cytoskeletal Proteins and Cytoskeletal-Associated Proteins
Several proteins which were identified as Nlk1 protein-interactants in the present invention were classified either as cytoskeletal proteins or proteins associated with cytoskeletal filaments. These aforementioned proteins include: (i) the skeletal muscle protein xcex1-tropomyosin, (ii) the intermediate filament vimentin and (iii) the desmosomal protein p0071 (which associates with intermediate filaments). Expressed sequences (hereinafter referred to as xe2x80x9cESTsxe2x80x9d) possessing homologies to cytoskeletal proteins or proteins associated with the cytoskeleton include: (i) the tropomyosin-homolog proteins encoded by IP-2 and IP-5 and (ii) the intermediate filament-associated protein encoded by IP-1.
The interaction between Nlk1 protein and cytoskeletal or cytoskeleton-associated proteins provides a link to hyperproliferative disorders, as it is known from prior art that the cytoskeleton undergoes extensive modification in those diseases. Cytoskeletal proteins provide the structural foundation which allows cells to exist in a highly organized manner. These cytoskeletal proteins not only maintain structural integrity, but might also be associated with signal transduction and suppression of tumorigenesis. Therefore, the interaction of Nlk1 protein with cytoskeletal or cytoskeleton-associated proteins may play a pivotal role in processes including, but not limited to, regulating tumor cell behavior during tumor development and metastasis, and neurodegenerative disorders. Furthermore, the interaction of Nlk1 protein with tropomyosin or tropomyosin-homolog proteins may also provide a link to cardiovascular diseases.
(i) xcex1-Tropomyosin
Human skeletal muscle a-tropomyosin (hTM; GenBank Acc. No. M19713; MacLeod and Gooding, 1988. Mol. Cell. Biol. 8:433-440) is a structural protein of muscle. The tropomyosins are highly conserved, coiled-coil actin-binding proteins found in most eukaryotic cells. The actin cytoskeleton is an intracellular structure, which is involved in the onset and control of cell shape and function. Actin filaments play important roles in mitosis, cell signaling, and motility and thus, the actin cytoskeleton is affected in many disease states. Striated and smooth muscle xcex1-tropomyosins differ as a consequence of alternative splicing of exons 2 and 9. See e.g. Ruiz-Opazo, et al., 1985. Nature 315:67-70. Non-acetylated, smooth tropomyosin binds actin with high affinity, whereas non-acetylated, striated tropomyosin requires the protein troponin, found only in striated muscle, for strong actin binding. See e.g., Hammell, et al., 1996. J. Biol Chem. 271:4236-4242.
Tropomyosin, in association with the troponin complexes (i.e., troponin-I, -T and -C) plays a central role in the Ca2+-dependent regulation of vertebrate striated muscle contraction. The function of this protein in smooth muscle and non-muscle cells remains unknown. Three distinct xcex1-tropomyosin messenger (mRNA) isoforms encode different protein isoforms; which are tissue-specific, developmentally regulated and (most probably) encoded by the same gene. Tissue-specific domains within these isoforms delineate the putative troponin-I and troponin-T binding domains of tropomyosin. See e.g., Ruiz-Opazo, et al., 1985. Nature 315:67-70.
Suppression of the specific muscle-type isoforms of tropomyosin is a common biochemical event in malignantly transformed cells. See e.g., Braverman, et al., 1996. Oncogene 13:537-545. For example, tropomyosins are commonly down-regulated in fibroblasts transformed by oncogenes and specific tropomyosin isoforms are down-regulated in human breast carcinoma cell lines. See e.g., Franzen, et al, 1996. Br. J. Cancer 73:909-913.
Additionally, mutations in the xcex1-tropomyosin gene are linked to familial hypertrophic cardiomyopathy (i.e., mutation Asp175Asn, Glu180Gly). Hypertrophic cardiomyopathy is a disease with an autosomal dominant pattern of heritability and is the most common cause of sudden-death in youth (see e.g., Palmiter and Salaro, 1997. Basic Res. Cardiol. 92:63-74; Kimura, et al., 1997. Nature Genet. 16:379-382; Davies and McKenna, 1995. Histopath. 26:493-500) and is characterized by left ventricular hypertrophy in the absence of an increased external load, as well as extreme myofibrillar disarray. Mutations in seven genes, all encoding sarcomeric proteins, have been identified as causes of familial hypertrophic cardiomyopathy. See e.g., Towbin, 1998. Curr. Opin. Cell Biol. 10:131-139. The genes include those encoding the cytoskeletal proteins such as xcex1-tropomyosin, xcex2-myosin heavy chain, cardiac troponin-T, myosin binding protein-C, myosin essential light chain, myosin regulatory light chain, and troponin-I.
Thus, xcex1-tropomyosin is involved in pathological processes including, but not limited to, tumorigenesis and hypertrophic cardiomyopathy.
(ii) IP-1 Protein
Within the present invention, IP-1 has been identified as a novel protein possessing homology to intermediate filaments associated proteins (e.g., the plectin proteins), which in-turn exhibit homologies to intermediate filaments (e.g., the keratin proteins). Thus, as discussed in the previous subsections, these aforementioned homologies serve to implicate IP-1 in various pathological processes including, but not limited to, tumorigenesis, neurodegenerative disorders and metabolic diseases.
(iii) IP-2 Protein
The Nlk1 protein-interacting protein (i.e., interactant), IP-2 (GenBank Acc. No. AA143467; Hillier, et al., 1997. Wash Univ.-NCI Human EST Project Proceedings) is an EST which, currently, has no published, ascribed biological function. However, as disclosed herein, the protein encoded by IP-2 is a xcex1-tropomyosin homolog protein, and accordingly, IP-2 may also play an important role in pathological processes including, but not limited to, the genesis of neoplasia and cardiovascular disorders.
(iv) IP-4 Protein
As disclosed herein, IP-4 has been characterized as a novel, Nlk1 protein-interacting protein (i.e., an interactant) of the present invention. IP-4 possesses homology to type-I collagen proteins, which have been shown to be involved in the regulation of a protease which has been broadly implicated in the neoplastic processes of invasion and metastasis within many cancer model systems including, but not limited to, human breast cancer (HBC). See e.g. Thompson, et al., 1994. Breast Cancer Res. Treat. 31:357-370. The adhesion of metastatic tumor cells to connective tissue elements (e.g., type I collagen) is required for the movement of these tumor cells into the subendothelial stroma and their subsequent growth at new locations. Accordingly, the type I collagen homolog protein IP-4 may be implicated in tumorigenesis and, particularly, in the metastatic dissemination of neoplastic cells.
(v) IP-5 Protein
The Nlk1 protein-interactant IP-5 possesses homologies to the amino-terminus of tropomyosin and to myosins. Furthermore, IP-5 has been shown to be a homolog of the Sry-like transcription factor, Sox 4, a DNA-binding protein. Accordingly, it is likely that the novel IP-5 protein is also, similarly involved in pathological processes including, but not limited to, the genesis of neoplasia and cardiovascular disorders.
(vi) Vimentin
Vimentin is a cytoskeletal protein which assembles into intermediate filaments (IFs) and belongs to the type III IF family (GenBank Acc. No. X56134; Honore, et al., 1990. Nuc. Acids Res. 18:6692-6698. IFs, as an intrinsic component of the cytoskeleton, control a large number and variety of biological events. Vimentin is expressed within central and peripheral neurons (i.e., neuronal progenitor cells, adult neurons, glial progenitor cells, mature astrocytes (see e.g., Ho and Liem, 1996. Cancer Metastasis Rev. 15:483-497) and in tumors of the nervous system. The expression of vimentin is tissue-specific and developmentally regulated and, therefore, vimentin serves as an efficacious marker for the determination of cell origin and the differentiation of the status of tumor cells.
When neoplastic transformation has taken place, affected cells reveal altered immunolocalization of IFs within the cytoplasm. See e.g., Hirasawa, et al., 1996. Nippon Rinsho 54:1542-1550. Vimentin has been associated with many tumors and it is a potential diagnostic immunohistochemical marker of soft tissue tumors. See e.g. Hibshoosh and Lattes, 1997. Sem. Oncol. 24:515-525. Additionally, in some cancers (e.g., malignant melanoma and breast carcinoma), vimentin and keratin IFs are co-expressed, thus lending credence to a putative role for vimentin in cancer progression. Over-expression of vimentin IFs in breast carcinoma leads to augmentation of motility and invasiveness which can be down-regulated by treatment with anti-sense oligonucleotides to vimentin, in vitro. See e.g., Hendrix, et al., 1996. Cancer Meta. Rev. 15:507-525.
In addition to vimentin""s role in tumorigenesis, the protein is also involved in all other cellular processes which are associated with IFs. For example, the rate of steroid synthesis is directly regulated by the rate of cholesterol transport to mitochondria which involves IFs, microfilaments and Ca2+/cahnodulin. Phosphorylation of IF-associated vimentin, causes breakdown of the IFs, which results in decreased cholesterol transport. See e.g., Hall, 1997. Steroids 62:185-189. Thus, vimentin functions in cell growth, differentiation, and other cytoskeletal finctions, and in tumorigenesis and metastatic spread, disorders of steroid metabolism, and disorders of cholesterol transport, including, atherosclerosis and cardiovascular disease.
Vimentin has also been shown to interact with: (i) the microfilament protein actin (see e.g., Ito, et al., 1996. Neurochem. Int. 29:383-389); (ii) the desmosomal plaque protein desmoplakin (see e.g., Meng, et al., 1997. J. Biol. Chem. 272:21495-21503) and (iii) the small heat-shock chaperone B-crystalline (see e.g., Djabali, et al., 1997. J. Cell Sci. 110:2759-2769).
Therefore, while it has been previously demonstrated that the actin cytoskeleton is affected in many disease states, quantification of the mechanisms by which altered structure or expression of actin, or of actin-binding proteins, cause specific defects are only now beginning to be elucidated.
(vii) p0071 Protein
The p0071 protein is a member of the Armadillo protein family (GenBank Acc. No. X81889; Hatzfeld and Nachtsheim, 1996. J. Cell Sci. 109:2767-2778), which are proteins associated with the cell-cell adherens junctional plaque. These proteins play an important role in cell-cell signaling processes through intercellular junctions. p0071 has been found to be expressed in all tissues thus far examined, and is localized at cell-cell borders and within the desmosomal plaque. p0071 may also be involved in regulating junctional plaque organization and the function of the adherin protein. See e.g., Hatzfeld and Nachtsheim, 1996. J. Cell Sci. 109:2767-2778.
The p0071 protein has been shown to be closely related to: (i) the murine p120 protein (a substrate of protein tyrosine kinase receptors) and (ii) the desmosomal band 6-protein (B6P)/plakoglobin. p0071 is a basic protein of 1,211 amino acid residues possessing a central xe2x80x9carmadilloxe2x80x9d repeat domain which is highly conserved between the p120, B6P/plakoglobin, and p0071 proteins. Desmosomal and adherens junction plaque proteins (e.g., plakoglobin) have been shown to exhibit remarkable efficiency in suppressing tumorigenesis. See e.g., Simcha, et al., 1996. J. Cell. Biol. 133:199-209.
In accord, p0071 has been implicated in cell-cell signaling, and thus, tissue-specific differentiation and development, as well as in tumorigenesis and metastatic dissemination of neoplastic tissue.
(B) Proteins Involved in Signal Transduction
In the present invention, three proteins, involved in signal transduction, are disclosed which were found to interact with Nlk1 protein. These proteins are: 14-3-3xcex5, TrkA and protein phosphatase-1xcex1. In addition, the novel protein IP-3, an ubiquitin-hydroxylase homolog, may also be implicated in similar physiological processes.
(i) 14-3-3xcex5
The highly conserved and ubiquitously expressed eukaryotic 14-3-3 family of proteins have been found to modulate a wide variety of cellular processes. Human 14-3-3xcex5 (GenBank Acc. No. U28936; Conklin, et al., 1995. Proc. Natl. Acad. Sci. USA 92:7892-7896) is an acidic, 30 Kdal isoform within 14-3-3 protein family. The 14-3-3 proteins are specific, phosphoserine-binding proteins (see e.g., Muslin, et al., 1996. Cell 84:889-897) which possess a highly conserved carboxyl-terminal domain (helices 7 and 8) through which the 14-3-3 proteins bind target proteins (see e.g., Ichimura, et al., 1997. FEBS Lett. 413:273-276).
The 14-3-3 family of proteins associate with a range of cellular or viral polypeptides which are involved in signal transduction, cell-cycle regulation and/or oncogenesis. See e.g., Aitken 1995. Trends Biochem. Sci. 20:95-97. 14-3-3 proteins have been demonstrated to interact with a wide variety of cellular proteins, including, but not limited to: (i) the serine/threonine protein kinase Raf-1 (see e.g., Zhang, et al., 1997. J. Biol. Chem. 272:13717-13724; Michaud, et al., 1995. MoL Cell. Biol.15:3390-3397); (ii) Bcr kinase (see e.g., Braselman and McCormick 1995. EMBO J. 14:4839-4848); (iii) protein kinase C (see e.g., Wheeler-Jones, et al., 1996. Biochem. J. 315:41-47) and phosphatidylinositol 3-kinase (see e.g. Bonnefoy-Berard, et al., 1995. Proc. Natl. Acad. Sci. USA 92:10142-10146), implicating 14-3-3 proteins in intracellular signal transduction and growth regulation.
For example, the 14-3-3 family of proteins may act as xe2x80x9cadapterxe2x80x9d proteins which modulate interactions between the components of signal transduction pathways; wherein the phosphorylation of the 14-3-3 proteins and/or their binding partners, may regulate these interactions. See e.g., Dubois, et al., 1997. J. Protein Chem. 16:513-522. 14-3-3 proteins are also known to interact with cdc25 phosphatase, which in turn activates the CDK proteins, which serve a critical function in cell-cycle progression. See e.g., Conklin, et al., 1995. Proc. Natl. Acad. Sci. USA 92:7892-7896. Furthermore, they interact with phosphorylated tryptophan hydroxylase in brain, thus implicating 14-3-3 proteins in serotonin biosynthesis and its role in neurological disorders and psychosis. See e.g., Banik, et al., 1997. J. Biol Chem. 272:26219-26225. The 14-3-3 family of proteins (including 14-3-3xcex5) are localized in the centrosome and spindle apparatus, and may function to xe2x80x9clinkxe2x80x9d mitogenic signaling, the cell-cycle and the centrosome duplication cycle. This intracellular localization is particularly interesting due to a recent experimental finding which links Nlk1 protein to the centrosome cycle. See e.g., Fry and Nigg, 1998. EMBO J. 18:179-186; Section 2(A), subsections i-vii, supra. More specifically, the 14-3-3xcex5 protein has been shown to interact with the central domain of the insulin receptor substrate (IRS-1) and with phosposerine residues contained within the carboxyl-terminus of the insulin-like growth factor I receptor (IGFIR). See e.g., Craparo, et al., 1997. J. Biol. Chem. 272:11663-11669; Furlanetto, et al., 1997. Biochem. J. 327:765-771. Therefore, 14-3-3xcex5 may function to regulate insulin signaling. See e.g., Ogihara, et al., 1997. J. Biol. Chem. 272:25267-25274.
Positionally, the 14-3-3xcex5 protein lies telometric to the Lis1 gene on chromosome 17p13.3 outside the Miller-Dieker syndrome chromosome region. See e.g. Chong, et al., 1996. Genome Res. 6:735-741. This chromosomal region is often associated with small deletions or translocations in a human developmental disease called isolated lissencephaly sequence disorder. See Hirotsune, et al., 1997. Genome Res. 7:625-634. Furthermore, this is a chromosomal region which is frequently deleted in several types of cancer, and thus, 14-3-3xcex5 may putatively function in a tumor suppressor capacity. For example, 14-3-3 proteins are expressed within lung cancer cells. See e.g., Setoguchi, et al., 1995. Hum. Anti. Hybrid. 6:137-144). The 14-3-3 proteins have also been demonstrated to be present in Alzheimer""s Disease neurofibrillary tangles. These proteins putatively affect MAP kinase signaling, resulting in tau protein hyper-phosphorylation, which in turn leads to the formation of the paired, helical filaments seen in the brains of individuals with Alzheimer""s Disease. See e.g., Layfield, et al., 1996. Neurosci. Lett. 209:57-60. Similarly, 14-3-3 proteins have been identified within the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease. See e.g., Rosenmann, et al., 1997. Neurol. 49:593-595.
Therefore, in conclusion, the 14-3-3 family of proteins are strongly implicated in various pathological disorders which include, but are not limited to, tumorigenesis and neurodegenerative disorders.
(ii) The Trk Oncogene (TrkA)
The human Trk oncogene (also known as the nerve growth factor receptor (TrkA); GenBank Acc. No. X03541; Martin-Zanca, et al., 1986. Nature 319:743-748)) belongs to the Trk family of tyrosine-protein kinases which function as signaling receptors that mediate the biological properties of the nerve growth factor (NGF) family of neutrophins. See e.g., Kaplan and Miller, 1997. Curr. Op. Cell. Biol. 9:213-221; Barbacid, 1995. Curr. Op. Cell. Biol. 7:148-155. TrkA, a protein of 641 amino acid residues, was initially isolated as a transforming oncogene in which most of the extracellular receptor domain is replaced by the coding sequence (i.e., the first 221 amino acid residues) of a tropomyosin-encoding gene. TrkA is primarily expressed in neuronal cells and mediates neurogenesis and survival of neurons. In addition, Trk receptors are implicated in the survival, differentiation, and growth of certain neurons and tumors of the nervous system. See e.g., Lucarelli, et al., 1995. J. Biol. Chen. 270:24725-24731. For example, human neuroblastoma (NB) cell lines have been shown to constitutively express low levels of TrkA mRNA, and such expression is associated with a favorable prognosis. See e.g., Tanaka, et al., 1995. Cancer 76:1086-1095.
Experimental evidence has demonstrated that the interaction of NGF with TrkA requires multiple contact sites involving at least five different domains of NGF. Additionally, phosphorylation of TrkA appears to be critical for the survival of neurons dependent upon NGF. See e.g., Nobes, et al., 1996. Neurosci. 70:1067-1079. In rodent models of diabetes there are marked deficits in the expression of NGF and its receptor TrkA. This deficient neurotrophic support generally leads to diabetic neuropathy. See e.g., Tomlinson, et al., 1996. Philos. Trans. R. Soc. Lond. B Biol. Sc. 351:455-462. In addition to their neurotrophic actions, NGF has been shown to exert specific effects on cells of the immune system, and TrkA has been found within cells of the immune system. See e.g., Burgi, et al., 1996. J. Immunol. 157:5582-5588; Ehrhard, et al., 1993. Proc. Natl. Acad. Sci. USA 90:5423-5427.
Other TrkA substrates include, but are not limited to: (i) phospholipase C (see e.g., Kaplan and Stephens, 1994. J. Neurobiol. 25:1404-1417); (ii) PI-3 kinase (see e.g., Holgado-Madruga, et al., 1997. Proc. Natl. Acad. Sci. U.S.A. 94:12419-12424); (iii) the adapter protein Shc (see e.g., Thomas and Bradshaw, 1997. J. Biol. Chen. 272:22293-22299); (iv) phosphotyrosine phosphatase SHP-2 (see e.g., Goldsmith and Koizumi, 1997. J. Neurochem. 69:1014-1019); (v) Ras GTPase activating protein (see e.g., Mattingly, et al., 1994. Mol. Cell. Biol. 14:7943-7952) and (vi) the mitogen-activated protein kinase ERKI (see e.g., Althaus, et al., 1997. J. Neurosci. Res. 50:729-742). It should be noted that most of these aforementioned proteins interact with different, conserved tyrosine residues within the carboxyl-terminal domain of TrkA (see e.g., Stephens, et al., 1994. Neuron 12:691-705; Obermeier, et al., 1994. EMBO J. 13:1585-1590), thus linking TrkA to several intracellular signaling pathways.
With respect to neuronal tissue, TrkA activity has been demonstrated to inhibit cell growth and neuritogenesis in PC12 pheochromocytoma and neuroblastoma cells (see e.g., Matsushima and Bogenmann, 1993. Mol. Cell. Biol. 13:7447-7456), wherein high expression of TrkA correlates with a generally favorable prognosis (see e.g., Hoehner, et al., 1995. Am. J. Path. 147:102-113). Similarly, NGF-stimulated TrkA activity also induced cellular apoptosis within 24 hours within medulloblastoma cells. See e.g., Muragaki, et al., 1997. J. Neurosci. 17:530-542. In Alzheimer disease, the number of neurons which express TrkA are markedly decreased in the nucleus basalis of Meynert as a consequence of cholinergic neuronal loss. See e.g., Boissiere, et al., 1996. Mol. Chem. Neuropathol. 28:219-223.
In addition to its presence in neuronal tissue, TrkA has also been demonstrated in a wide range of non-neuronal tissues. TrkA was originally identified in a colon carcinoma (see e.g., Mitra, et al., 1991 Oncogene 6:2237-2241), and subsequently, this transforming oncogene has been detected at various levels in all tissues examined thus far, except for the heart and liver (see e.g., Shibayama and Koizumi, 1996. Am. J. Pathol. 148:1807-1818). For example, mutations within the TrkA oncogene are frequently found in human thyroid tumors (see e.g., Salvatore, et al., 1996. Eur. J. Endocrinol. 134:177-183) and TrkA has also implicated in the transformation of the human melanoma cell line XP44RO (see e.g., Lafarge-Frayssinet, et al., 1995. Anticancer Res. 15:1205-1213).
Hence, the TrkA transforming oncogene has been implicated in the physiological processes of cell growth, survival and apoptosis, neurogenesis, neuritogenesis, immune system, secretion, and intracellular signal transduction. In addition, it has also been implicated in the pathophysiology of tumorigenesis, metastatic spread, Alzheimer""s disease and neurodegeneration, diabetes, and disorders of the immune system.
(iii) Protein Phosphatase 1xcex1 (PP1xcex1)
Protein phosphatase la (PPla; GenBank Acc. No. M63960; Song, et al., 1993. Gene 129:291-295) is a 330 amino acid residue, serine/threonine-specific, protein phosphatase which affect a wide variety of important signal transduction processes, including cell-cycle progression, T-cell activation and neurotransmitter receptor activity.
An isoform of PPla has been shown to associate with retinoblastoma protein during the M to G1 transition. See e.g., Durfee, et al., 1993. Genes Dev. 7:555-569. Additionally, PP1xcex1 interacts with the cellular oncogene Hox11 (an orphan homeobox gene which controls the genesis of the spleen and liver) which was originally isolated from a chromosomal breakpoint in human T-cell leukemia. Hox11 targets both PP1xcex1 and PP2xcex1, which in turn are targets for oncogenic viruses and chemical tumor promoters. See e.g., Kawabe, et al., 1997. Nature 385:454-458. It has been hypothesized that inhibition of PP1xcex1 and PP2xcex1 by Hox 11 abrogates a G2 checkpoint, thus promoting genomic instability and oncogenesis. Similarly, in fission yeast, PP1xcex1-like phosphatases have been shown to inhibit the cell-cycle protein p34cdc2/cyclin kinase and thereby negatively-regulate entry into mitosis. See e.g. Yanagida, et al., 1992. Ciba Found. Symp. 170:130-140.
PP1xcex1 is regulated by several inhibitory proteins (e.g., the dopamine- and cAMP-regulated phosphoprotein (DARPP-32); PPI-inhibitor-1; PPI-inhibitor-2 and the like) which inhibit by binding to the catalytic domain (amino acid residues 7-300). The protein is also inhibited by various toxins and tumor promoters (e.g., clayculin A, okadaic acid, dinophysitoxin 1, microcystin, nodularin, tautomycin and cantharidic acid) by interacting at a common inhibitor site on PP1xcex1. See e.g., Huang, et al., 1997. Proc. Natl. Acad. Sci. USA 94:3530-3535; Fujiki and Sugamura, 1993. Adv. Cancer Res. 61:143-194).
The nuclear translocation of PP1xcex1 has also been implicated in monocyte differentiation (see e.g., Omay, et al., 1995. Cancer Res. 55:774-780), probably by effecting the relocation of associated proteins. Nuclear translocation has also been associated with a malignant phenotype in hepatomas. See e.g., Saadat, et al, 1995. Cancer Lett. 94:165-170. The inhibition of PP1xcex1, which is expressed in high levels within mammalian brain, has been associated with the stimulated secretion of the Alzheimer amyloid precursor protein (APP), thus diverting the processing of APP to non-amyloidogenic production of the large extracellular domain APPS. The aberrant processing of the Alzheimer amyloid precursor protein, and its amyloidogenic A-xcex2, fragment, is thought to be centrally involved in Alzheimer""s disease See e.g., de Cruz e Silva, et al., 1995. Mol. Med. 1:535-541.
In addition, the catalytic subunit of rabbit muscle PP1xcex1 has been found to bind to the glycolytic enzyme muscle phosphofructokinase (see e.g., Zhao and Lee, 1997. Biochem. 36:8318-8324) indicating that PP1xcex1 can control glycolytic flux by protein phosphorylation and/or dephosphorylation. Interestingly, in addition to the glycogen-bound form of PP1xcex1, a myosin-bound form of PP1xcex1 exist in skeletal and cardiac muscles which dephosphorylates myosin and the PP1xcex1 catalytic subunit has been shown to be highly enriched in isolated rat postsynaptic densities. See e.g., Chisholm and Cohen, 1988. Biochem. Biophys. Acta 971:163-169.
Four major rat brain PP1xcex1xcex1-binding proteins (with molecular weights of 216, 175, 134, 75 Kdal) have been characterized. See e.g., Colbran, et al., 1997. J. Neurochem. 69:920-929. As disclosed herein by the present invention, the dephosphorylation of rabbit brain tryptophan hydroxylase by PP1xcex1 can be inhibited by 14-3-3 proteins. See Section 2(B).
Therefore, in conclusion, PP1xcex1 has been implicated in pathological disorders including, but not limited to, tumorigenesis, neurodegenerative disorders, and metabolic disorders.
(iv) IP-3 Protein
IP-3 is a novel protein which has been shown to possess homology to ubiquitin-specific hydrolases. Therefore, IP-3 putatively encodes a protein which belongs to the ubiquitination/deubiquitination system which controls the degradation of many cellular proteins. See e.g., Kalderon, 1996. Curr. Biol. 6:662-665. The ubiquitinated proteins were initially found within neurons in several major human neurodegenerative diseases (e.g. Alzheimer""s disease, diffuse Lewy body disease, motor neuron disease and the like). See e.g. Mayer, et al., 1991. Acta Biol. Hung. 42:21-26. There is also more recent evidence regarding a putative role for the members of the ubiquitin hydrolase family playing a role in oncogenesis. See e.g., Swanson, et al., 1996. Hum. Mol. Genet. 5:533-538. Specifically, ubiquitin carboxyl-terminal hydrolase MRNA was detected in a tumor-derived neuroblastoma cell line (Neuro-2a), in non-neuronal, acute lymphoblastic leukemia (see e.g., Maki, et al., 1997. Biotech. Histochem. 72(1):38-44), in undifferentiated embryonic neoplasms (e.g., primitive neuroectodermal tumors, medulloblastomas and in oat cell carcinomas of the lung; see e.g., Ermisch and Schwechheimer, 1995. Clin. Neuropathol. 14(3): 130-136). Accordingly, the presence of this protein could be utilized as a sensitive marker of these aforementioned tumor groups.
(C) DNA-Binding Proteins
As previously discussed, the Nlk1 protein is involved in chromosomal condensation. See Section 2(A). In the present invention, one DNA-binding protein was demonstrated to interact with Nlk1 proteinxe2x80x94the integrase interactor 1 protein. However, recent experimental evidence has also shown a putative interaction of the Nlk1 protein with the newly discovered human nuclear protein HEC (see PCT Publication WO 98/27994).
(i) Integrase Interactor 1 Protein (Ini-1)
The integrase interactor 1 (Ini-1) cellular protein has been found to interact with, and activate human immunodeficiency virus (HIV-1) integrase. Ini-1, the human homolog to the yeast transcription faction SNF5 (see e.g., Kalpana, et al., 1994. Science 266:2002-2006), is part of a protein complex which is thought to alter nucleosomal structure and thus, influence the selection of site(s) for HIV-1 genomic integration. See e.g., Miller and Bushman, 1995. Curr. Biol. 5:368-370.
The early stages of HIV-1 replication involve reverse transcription of the viral RNA and integration of the resulting cDNA into the host genome. HIV-1 cDNA integration requires the binding of the human DNA prior to the connection of the viral and host DNAs. See e.g., Miller, et al., 1995. Curr. Biol. 5:1047-1056. Recent evidence suggests that the retroviral integrase protein Ini-1 functions as a nuclear factor which promotes the targeting and insertion of the viral DNA into host chromosomal targets. Conserved domains of Ini-1 (i.e., amino acid residues 220-270) are critical for the host DNA-binding and integration of the retroviral cDNA. See e.g., Drelich, et al., 1993. J. Virol. 67:5041-5044; Eijkelenboom, et al., 1995. Nat. Struct. Biol. 2:807-810. As the integration of viral DNA into the human genome is required for replication of HIV-1, Ini-1 is logical target for anti-retroviral strategies.
Ini-1 has also been shown to interact with the nuclear antigen 2, a viral nuclear protein expressed in latently infected B lymphocytes essential to the immortalization of B cells by the Epstein-Barr virus (EBV). See e.g., Wu, et al., 1996. J. Virol. 70:6020-6028. Thus, Ini-1 is also strongly implicated in the process of DNA viral infection.
As previously stated, the newly discovered human nuclear protein HEC (see PCT Publication WO 98/27994)HEC is believed to interact with other proteins which serve an important role in mitosis (e.g., Nlk1, sb1.8 and the like) through a leucine heptad repeat domain. HEC appears to be important in chromosomal segregation during M phase, as the highest levels of expression were found in rapidly-dividing cells (e.g., neoplastic cells) were it was localized to centromere during mitosis. Additionally, HEC inactivation was demonstrated to result in disordered sister chromatid alignment and separation.
In brief, the Nlk1 protein has been demonstrated to form complexes, which heretofore have not been described, with the following cellular proteins: TrkA, protein phosphatase 1xcex1, 14-3-3xcex5, xcex1 tropomyosin, vimentin, p0071, Ini-1, IP-1, IP-2, IP-3, IP-4 and IP-5. In genes which encode the IP-1, IP-2, IP-3, IP-4 and IP-5 proteins have not been previously described.
The present invention discloses herein compositions and methodologies for the production of protein complexes comprised of the Nlk1 protein and various other proteins which interact with (i.e., bind to) said Nlk1 protein. The proteins which have been demonstrated to form complexes with the Nlk1 protein will be designated hereinafter as xe2x80x9cNlk1 protein-IPxe2x80x9d for Nlk1 protein interacting protein; whereas a complex of the Nlk1 protein and a Nlk1 protein-IP will hereinafter be designated as xe2x80x9cNlk1 proteinxe2x80xa2Nlk1 protein-IPxe2x80x9d.
More specifically, the present invention relates to complexes of the Nlk1 protein, and derivatives, fragments and analogs thereof, with the following cellular proteins:
(i) TrkA; (ii) protein phosphatase 1xcex1; (iii) 14-3-3xcex5; (iv) xcex1 tropomyosin; (v) vimentin; (vi) p0071; (vii) Ini-1; (viii) IP-1; (ix) IP-2; (x) IP-3; (xi) IP-4 and (xii) IP-5, as well as their derivatives, analogs and fragments.
Methods of production of the Nlk1 proteinxe2x80xa2Nlk1 protein-IP complexes, and derivatives and analogs of these aforementioned proteins and protein complexes by, for example, recombinant means, will also be disclosed herein. Various pharmaceutical compositions relating to the Nlk1 protein:Nlk1 protein-IPs, Nlk1 proteinxe2x80xa2Nlk1 protein-IP complexes, and derivatives, fragments and analog thereof, will also be disclosed by the present invention.
The present invention will further provide methodologies for the modulation (i.e., inhibiting or enhancing) of the activity of the Nlk1 proteinxe2x80xa2Nlk1 protein-IP complexes, particularly: the following complexes: Nlk1 proteinxe2x80xa2TrkA; Nlk1 proteinxe2x80xa2xcex1 tropomyosin; Nlk1 proteinxe2x80xa2vimentin; Nlk1 proteinxe2x80xa2p0071; Nlk1 proteinxe2x80xa2protein phosphatase 1xcex1; Nlk1 proteinxe2x80xa214-3-3xcex5; Nlk1 proteinxe2x80xa2Ini-1; Nlk1 proteinxe2x80xa2IP-1; Nlk1 proteinxe2x80xa2IP-2; Nlk1 proteinxe2x80xa2IP-3; Nlk1 proteinxe2x80xa2IP-4 and Nlk1 proteinxe2x80xa2IP-5. The protein components of these aforementioned complexes have been implicated in a plethora of cellular and physiological processes, including, but not limited to: (i) control of cell-cycle progression; (ii) cellular differentiation and apoptosis; (iii) regulation of transcription; (iv) control of intracellular signal transduction and (v) pathological processes including, but not restricted to, hyperproliferative disorders (e.g., tumorigenesis and tumor progression); neurodegenerative diseases; cardiovascular disease; metabolic diseases and viral infections.
Accordingly, the present invention provides methodologies for the screening of Nlk1 proteinxe2x80xa2Nlk1 protein-IP complexes, particularly complexes of the Nlk1 protein with TrkA, protein phosphatase 1xcex1, 14-3-3xcex5, xcex1 tropomyosin, vimentin, p0071, Ini-1, IP-1, IP-2, IP-3, IP-4 and IP-5, as well as derivatives, fragments and analogs thereof, for the ability to modulate or alter cell functions, particularly those cell functions in which Nlk1 protein and/or a Nlk1 protein-IP has been implicated including, but not limited to: control of cell-cycle progression; cellular differentiation and apoptosis; regulation of transcription; control of intracellular signal transduction; and pathological processes including, but not restricted to, hyperproliferative disorders (e.g., tumorigenesis and tumor progression); neurodegenerative diseases; cardiovascular disease; metabolic diseases viral infections.
The present invention further relates to therapeutic and prophylactic, as well as diagnostic, prognostic and screening methodologies and pharmaceutical compositions which are based upon Nlk1 proteinxe2x80xa2Nlk1 protein-IP complexes (and the nucleic acids encoding the individual proteins constituents which participate in the complexes). Therapeutic compounds of the invention include, but are not limited to: (i) Nlk1 proteinxe2x80xa2Nlk1 protein-IP complexes, and complexes where one or both members of the complex is a derivative, fragment or analog of the Nlk1 protein or a Nlk1 protein-IP; (ii) antibodies to, and nucleic acids encoding the foregoing and (iii) antisense nucleic acids to the nucleotide sequences encoding the various protein complex components. Diagnostic, prognostic and screening kits will also be provided.
Animal models and methodologies of screening for various modulatory agents (i.e., agonists, antagonists and inhibitors) of the activity of the Nlk1 protein:Nlk1 protein-IPs and Nlk1 proteinxe2x80xa2Nlk1 protein-IP complexes, are also disclosed herein.
Methodologies for the identification of molecules which inhibit, or alternatively, which increase the formation/synthesis of the Nlk1 protein:Nlk1 protein-IPs and Nlk1 proteinxe2x80xa2Nlk1 protein-IP complexes will also be provided by the present invention.